Multi-bioreactor hollow fiber cell propagation system and method

ABSTRACT

A unique hollow fiber multiple bioreactor system and method for the propagation of cells and the production of various cell propagation products is described. Simultaneous fluid flow through lumens of hollow fibers in bioreactors is maintained constant across multiple bioreactors so production is maintained essentially constant. The system and method include use of a flow block holder which allows a dissolved oxygen probe to be calibrated at the same pressure as the media in the bioreactor system.

This is a continuation-in-part application of application Ser. No. 07/480,533 filed Feb. 15, 1990, now U.S. Pat. No. 5,126,238.

FIELD OF THE INVENTION

This invention relates to a system and a method for the propagation of cells and to the production of various cell propagation products. More particularly, the invention relates to the propagation of cells in a unique hollow fiber bioreactor system and process.

BACKGROUND OF THE INVENTION

The propagation of suspension and anchorage dependent cells in hollow fiber bioreactors is variously described in the prior art. In general, known procedures entail the use of bioreactors comprising a plurality of media permeable parallel hollow fibers surrounded by an extracapillary space (ECS). Cell growth medium passed through the hollow fiber lumens permeates the lumen walls to support cell growth in the ECS. See, e.g. U.S. Pat. Nos. 3,821,087; 4,439,322 and Ramsay et al. In Vitro 20:10 (1984).

SUMMARY OF THE INVENTION

This invention provides a unique hollow fiber cell propagation system and process. The cell propagation system is compact and highly efficient. It includes a plurality of novel elements including a bioreactor, or bioreactors, having hollow fibers of unique composition and interrelated means to control dissolved oxygen, basal medium and reagent composition, temperature, pH and waste disposal means.

The process as practiced in the system of the invention provides high cell and product yields with a low consumption of medium and nutrients. These benefits are attributable to a plurality of innovations which individually and in combination provide for the uniquely efficient propagation of suspension and anchorage dependent mammalian, insect, avian, plant and fungal cells and associated products.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the cell propagation system of the invention.

FIG. 2 is a sectional view of a dissolved oxygen probe and probe block useful in the system of the invention.

FIG. 3 is a schematic view of a means, useful in the invention for control of the temperature of the basal medium and reagents utilized in the system of the invention.

FIG. 4 is a schematic view of a means useful to control the composition of the basal medium and reagents utilized in the system.

FIG. 5 is a schematic view of a waste disposal system useful in the invention.

FIG. 6 is a schematic view of an oxygenator useful in the system of the invention.

FIG. 7 is a sectional view of a loop heater useful in the system of the invention.

FIG. 8 is a schematic illustration of an integrated, compact configuration of a single bioreactor system which embodies the invention; elements shown in phantom by broken lines are positioned behind elements shown in solid lines; all elements are directly or indirectly mounted on a transparent polycarbonate mounting sheet.

FIG. 9 is a graph which illustrates the parallelism between GUR and .increment.DO in a relatively aerobic system run.

FIG. 10 is a graph which illustrates the gradual decrease in aerobic efficiency of a bioreactor during a 110 day system run.

FIG. 11 is a schematic view of a multiple bioreactor recirculation loop configuration.

FIG. 12 is a schematic view of a multiple bioreactor showing an extracapillary space nutrient delivery and harvest configuration.

DETAILED DESCRIPTION OF THE INVENTION

In the specification, the following symbols have the indicated significance or meaning:

    ______________________________________                                         DO1          Dissolved oxygen value upstream of                                             bioreactor(s), expressed as % (percentage                                      of air saturation)                                                DO2          Dissolved oxygen value downstream of                                           bioreactor(s), expressed as % (percentage                                      of air saturation)                                                ΔDO    Dissolved oxygen differential:                                                 ΔDO = DO1 - DO2                                             ECS          Extracapillary space                                              Gi           Glucose concentration entering system,                                         expressed in mg/l                                                 Go           Glucose concentration exiting system,                                          expressed in mg/l                                                 GUR          Glucose utilization rate, expressed in                                         mg/hr Calculated as:                                                            ##STR1##                                                                      where v = basal medium feed rate                                  Basal Medium Rate at which basal medium and reagents                           Feed Rate    enter and leave the system. Expressed in                                       ml/hr                                                             Lumen (or Loop)                                                                             Space inside a hollow fiber                                       Basal Medium Nutrient medium comprises water, salts,                                        amino acids, vitamins.                                            Reagents     Substances added to a medium and known to                                      have a particular action under certain                                         conditions                                                        Plateau Section                                                                             Portion of hollow fiber run at which                                           process conditions are stable                                     Growth Factor                                                                               Combination of defined basal medium                               Mixture      growth factors and/or serum                                       System Cancellation                                                                         Time at which growth support for the                                           hollow fiber system is terminated                                 ______________________________________                                    

In general, the cell propagation process of the invention may include some or all of the steps 1 to 14 described hereinafter.

1. Cell lines are grown in several different basal medium and reagent types and combinations in order to select the most appropriate growth medium for the hollow fiber run. The selected growth medium is reduced to the simplest practical form.

2. Following hollow fiber system sterilization and assembly, basal medium is fed into the system and temperature control is set at about 37° Celsius.

3. Target Go values, preferably in a range of 50-500 mg/l and GUR (range is product and cell line dependent) values are selected for the plateau section of the run.

4. Gas composition (CO₂, N₂, air) is selected for system start up based upon pH requirements and cell line sensitivity to O₂. These gases are blended and humidified to approximately 100% relative humidity before delivery to the oxygenator.

5. Loop circulation rates are established which will support bioreactor oxygen demands generated by targeted GURs. DO2 values are preferably held above 10% saturation of air by lumen flow selection.

6. ECS growth factor mixture is formulated based upon historical experimentation. ECS growth factor delivery and product harvest program is developed as a function of targeted GUR, feedback inhibition data, cell line and product requirements. ECS growth factor delivery and product harvest program is entered into the programmable controller.

7. Basal medium feed rate is set by adjusting the basal medium feed pump. The initial feed rate may be set high (in excess of that required for the targeted GUR) to provide accelerated generation of cell mass.

8. A cell inoculum is introduced into the ECS of the bioreactor(s). Cell count of the inoculum is cell line and bioreactor surface area dependent.

9. As target Go and GUR are approached, basal medium feed rates are reduced step-wise until target Go and GUR values are reached substantially simultaneously.

10. CO₂ flows are gradually reduced as target GURs are reached to maintain pH values in preferred ranges.

11. Productivity (protein production) is correlated with .increment.DO values.

12. GURs are held constant by fine adjustments in basal medium feed rate and system reagent composition.

13. Aerobic efficiency of the system is monitored by the ratio of GUR/.increment.DO and as it relates to product output. System cancellation is determined by comparing these values to established limits developed through production cost analysis.

14. Data generated by the run may be added to the general data base and used to further define parameters such as optimal operational GURs, .increment.DOs, ECS programming (growth factor delivery frequency, product harvest frequency and volume) and reagent recipes in future runs.

The system in which this process may be practiced is schematically illustrated by FIGS. 1 to 7 and 11 to 12.

THE SYSTEM OF THE INVENTION

As shown in FIG. 1, the system includes interconnected ECS product/feed section 100, bioreactor section 200, loop section 300, loop feed/waste section 400, oxygenator section 500, gases section 600 and electronics section 700. The system preferably takes the compact form shown by FIG. 8 in which various of the system elements are mounted on a plastic, preferably transparent polycarbonate mounting sheet. As shown in FIG. 1, the bioreactor section 200 includes one bioreactor 201. This arrangement can be expanded with multiple bioreactors 201 as shown in FIGS. 11 and 12.

The ECS Product/Feed Section

The ECS product/feed section 100 includes an ECS feed reservoir 101, an ECS product reservoir 102, and an ECS pump 103. ECS feed reservoir 101 is connected by line 104 to the pump 103, and the bioreactor section 200. ECS product is connected by lines 105 to the pump 103 and the bioreactor section 200.

The BioReactor Section 200

As shown in FIG. 1, the bioreactor section 200 includes one hollow fiber bioreactor 201 connected through lines 104 and 105 to the ECS product/feed section 100 and through lines 202 and 203 to the loop section 300. The bioreactor 201 is provided at each end with manifolds 204 and 205 to which lines 202 and 203 are connected.

In order to increase cell production hollow fiber surface area can be increased by adding bioreactors to the system. Increasing the number of bioreactors, however, results in proportional increases in system complexity and a tendency to lose cell mass homogenity because of unequal medium flow in both the ECSs and lumens of various bioreactors. Uneven medium flows cause not only unequal distribution of growth nutrients but also unequal product harvesting. These problems are overcome by the multiple bioreactor section of the present invention through use of unique configurations for medium flow paths and employment of unique system components. Further, the system configuration of the present invention is not limited to either an even or odd number of bioreactors, but can be applied to various numbers of bioreactors in parallel.

The recirculation loop configuration of the present invention for multiple bioreactors is shown in FIG. 11. This recirculation loop configuration consists of a manifold 211 which is designed to split flows from a single loop stream, i.e., lines 202 and 203, into multiple loop streams required in such a number as to service multiple bioreactors 201 arranged in parallel with respect to each other. The manifold 211 is configured such that a single stream enters and a single stream exits. The single entrance stream and single exit stream provide fluid flow at diagonally opposite points to the manifold 211. Such an arrangement of equal size bioreactors 201 promotes nearly equal flow rates through lumens of each bioreactor 201 and therefore promotes equal metabolic activity in each bioreactor 201 which in turn results in equal product formation in each bioreactor 201. As with the single bioreactor 201 system shown in FIG. 1, dissolved oxygen measurements are made upstream of line 203 and downstream of line 202 in the loop section 300 described below.

Designs as described for loop flow manifolds 211 for multiple bioreactors 201 have little utility for the ECS flow system for multiple bioreactors 201 due to low flow rates and also low ECS pressures which create poor flow distribution patterns. A unique non-invasive pinch valve system and manifold is used in the present invention to effectively isolate each bioreactor 201 in the parallel configuration so that delivery of growth factor mixture and harvesting of ECS product is accurate in volume and specific for each bioreactor 201. Interreactor 201 flow is prevented on the upstream side by the presence of ECS pinch valves and on the downstream side by the presence of equal transmembrane pressures in each bioreactor 201.

A programmable controller 701, as shown in FIG. 12, operates through the broken lines on ECS pump 103 in conjunction with a battery of pinch valves 210, 212, 214 and 216 at a desired frequency. The ECS pump 103 delivers growth factor mixture to the upstream section, at which point flow is possible only through an open pinch valve 210, 212, 214 or 216. The method of ECS operation occurs as follows. The programmable controller 701 activates the ECS pump 103 and pinch valve 210 simultaneously. Then growth factor mixture flows from the ECS feed reservoir 101, through the ECS pump 103, into line 104 and then into the upstream section of the first bioreactor 201. Because only pinch valve 210 is open, the only available flow path is through the first bioreactor 201. Product exits the first bioreactor 201 into line 105, through the ECS pump 103 and into the ECS product reservoir 102. The system is configured so that ECS pump 103 acts as a shut off valve on the downstream side of the bioreactors 201 when the ECS pump 103 is not activated, thus preventing transmembrane flow of media from the loop into the ECS product reservoir 102. After the first bioreactor 201 has completed its nutrient feed and product collection cycle, pinch valve 210 is deactivated and pinch valve 212 is immediately activated. During this transition, the ECS pump 103 continues to operate. The above described steps are repeated in a sequential fashion for the remaining bioreactors 201. After sequential nutrient feeding and product collection for each bioreactor 201, the ECS system remains at rest until the programmable controller 701 begins the series of operations again.

In order to accomplish the operation described above, the ECS pump 103 is peristaltic in operation, i.e., continuous contraction waves caused by the ECS pump 103 act on lines 104 and 105 to force contents onward. The peristaltic ECS pump 103 is arranged to have both lines 104 and 105 in the same ECS pump 103 at the same time so both lines 104 and 105 are acted on at the same time. Both lines 104 and 105 are of the same outside and inside diameter and consequently have the same flow rates. Finally, the ECS valves 210, 212, 214 and 216 are normally closed unless energized to open by a signal from the programmable controller 701.

The Loop Section 300

The loop section 300 is connected through: (1) lines 202 and 203 to the bioreactor section 200; (2) lines 304 and 305 to the loop feed waste section 400; and, (3) lines 306 and 307 to the oxygenator section 500. The loop section 300 includes two dissolved oxygen probes 308 and 309 with associated dissolved oxygen probe flow blocks 310 and 311, a loop heater 312, a loop flowmeter 313, a loop sampler 314, a loop reservoir 315, a loop circulation pump 316, and a pH probe 332 and pH probe flow block 317.

The dissolved oxygen flow block 310 is connected by line 202 to the bioreactor section 200 and by line 318 to the loop heater 312 which is connected by the line 319 to the loop flowmeter 313. The flowmeter is connected by line 320 to the loop reservoir 315. The line 320 is fitted with a loop sampler 314.

Loop reservoir 315 is connected by line 321 to loop circulation pump 316. The loop section 300 is connected through lines 306 and 307 to the oxygenator section 500. A pH probe 332 and pH probe flow block 317 are located in line 322.

The dissolved oxygen probe flow block 311 is connected to the bioreactor section 200 by the line 203 and by the line 323 to the resistance temperature detector (RTD) probe flow block 324 having an associated RTD probe 325. The RTD probe flow block 324 is connected by the line 307 to the oxygenator section 500.

The Loop Feed/Waste Section 400

The loop feed/waste section 400 is connected by the lines 304 and 305 to the loop section 300. It includes a waste reservoir 401 connected to the loop reservoir 315 through the line 304; a basal medium feed reservoir 402 connected to the line 305 through the loop feed pump 403; and a glucose reservoir 404 connected by the line 406 through the glucose feed pump 405 to the line 305.

The Oxygenator Section 500

The oxygenator section 500 is connected by lines 306 and 307 to the loop section 300 and by the line 501 to the gases section 600. The oxygenator section 500 includes a hollow fiber oxygenator 502 having manifolds 504 and 505. The manifold 505 is connected to the line 307 from the loop section 300. The line 306 is fitted with a loop filter 506 positioned before the manifold 504. Oxygenator exhaust line 507 is provided with an exhaust filter 508.

The Gases Section 600

The gases section 600 is connected to the oxygenator section 500 by the line 501. It includes a source of nitrogen, e.g., a cylinder 601, a source of carbon dioxide, e.g., a cylinder 602, and an air pump 603, each connected to manifold line 604 by lines 605, 606 and 607, respectively. Each of lines 605, 606 and 607 is fitted with an oil/particle gas filter 608 and a gas flowmeter 609.

Manifold line 604 is connected through line 610 to gas humidifier 611. The line 610 has an in-line gas feed filter 612. A humidifier water fill line 614 with an in-line filter 613 is attached to line 610.

The Electronics Section 700

The electronics section 700 includes a programmable controller 701, dissolved oxygen monitors 702, a temperature monitor and controller 703, a pH monitor 704, a loop circulation pump controller 705, and a basal medium feed pump controller 706. These electronic components are functionally interconnected with relevant sections of the system as shown by the broken lines in FIGS. 1 and 12.

The electronic elements, per se, are commercially available items. The component interconnections are made in a known manner and can readily be accomplished by the skilled person.

DETAILED DESCRIPTION OF CERTAIN SYSTEM ELEMENTS

Except as specifically described herein, the various elements of the system depicted by FIGS. 1, 11 and 12 are of conventional design.

1. The Hollow Fiber Bioreactor 201

Hollow fiber bioreactors useful in the invention may be of known design. See, e.g., U.S. Pat. Nos. 4,804,628; 3,821,087; and 4,391,912.

A preferred hollow fiber membrane and bioreactors including such hollow fiber membranes are described in co-pending application of Gebhard and Veeramallu, Ser. No. 355,590, filed Jun. 3, 1989. Permeable membranes useful in the bioreactors of this invention may be selected to accommodate the particular cell propagation process or method in which the bioreactor is to be used. Various types of semi-permeable membranes are known. Such semi-permeable membranes may be cellulosic or of synthetic origin. Polysulfone, polymethyl methacrylate and blends of isotactic and syndiotactic polymethyl methacrylate are appropriate. Semi-permeable hollow fibers of the kind described in U.S. Pat. No. 3,896,061 incorporated herein by reference are appropriate.

A preferred form of hollow fiber bioreactor is a dialysis device sold by Toray Industries, Tokyo, Japan.

2. The Dissolved Oxygen Probe and Probe Flow Block

FIG. 2 schematically illustrates a dissolved oxygen probe 308 and associated flow block 310. As shown, these components include the dissolved oxygen probe 308 and the flow cell body 310 which functions as a flow block. Medium from the bioreactor section 200 enters the flow cell body 310 through the line 202. Fluid exits through the line 318. The dissolved oxygen probe 308 is fitted to the body 310 by the O-ring 333. A teflon membrane 329 is fitted to the distal end of the probe 308. An observation window 330 fitted with an O-ring seal 326 is provided. The annular space 327 between the probe 308 and the body 310 is provided with an appropriate, e.g., 0.2 μm, filter 328.

The dissolved oxygen probe and associated flow block permits on-line measurement of the dissolved oxygen content of the media. As shown in FIG. 1, one such device 309, 311 is positioned to measure the DO1 of the media prior to entry into the bioreactor 201 and a second such device 308, 310 measures media DO2 after passage through the bioreactor(s). In use, an air bubble is introduced sterilely through the filter 328 to displace medium below the teflon membrane 329 of the dissolved oxygen (DO) probe 308. The loop flow channel is arranged such that flow continues through the flow cell, as shown in FIG. 2, through lines 202, the flow cell and line 318. Because the medium flow is maintained during introduction of the air bubble, the pressure in the bubble is consistent with that of the medium flowing through the loop.

Upon completion of the calibration, the air bubble is removed through the filter 328 and the filter sealed closed. The observation window 330 is provided to permit monitoring of the entire calibration process and of the integrity of the DO probe membrane 329.

Commercially available DO probes may be utilized. A preferred galvanic dissolved oxygen probe is available from Phoenix Electrode, 6103 Glenmont, Houston, Tex. 77081, as item 025NG-15-80. The DO probe is appropriately connected to the electronic DO monitor 702.

Aerobic Efficiency

Aerobic efficiency is determined by simultaneously measuring the GUR of the bioreactor(s) and the dissolved oxygen differential (.increment.DO) across the bioreactor(s) and using the value GUR/.increment.DO to assess the performance of the bioreactor(s) in terms of product formation; product formation and GUR/.increment.DO relationships having been established through historical performance. For these values to have significance, other system operating parameters must be taken into account such as loop circulation rate, basal medium type, temperature and Go. For example, using a murine hybridoma system as a model with a loop circulation rate of 350 ml/min, Dulbecco's Modified Eagles high glucose [4500 mg/l] medium as the basal medium, a temperature of 37° C., and a Go value in the range of 200 to 300 mg/l, GUR/.increment.DO values of 3 to 6 (mg/hr)/% air saturation indicate an aerobically efficient bioreactor with acceptable product output while GUR/.increment.DO values of 10 to 12 (mg/hr)/% air saturation indicate a bioreactor with significant oxygen mass transfer problems, low productivity and potential for system cancellation.

Real time verification of cell metabolism in the bioreactor(s) is provided by using the .increment.DO value as an indication of the oxygen consumed in the process of respiration. This method of metabolism verification is of exceptional value when a continuity of .increment.DO has been established. Deviations to the established .increment.DO continuity enable the system operator to immediately take appropriate corrective action without reconducting time consuming off line assays.

3. The Hollow Fiber Temperature Control System

FIG. 3 is a schematic depiction of a preferred hollow fiber temperature control system useful in the invention.

As shown in schematic FIG. 3, a hollow fiber bioreactor 201 is connected by the line A to the loop heater 312 which is connected by broken line B to the electronic temperature monitor and controller 703. A thermostat 331 is fitted between the heater 312 and the line B.

The heater 312 is connected by the line C to the loop reservoir 315 which is connected by line D to the resistance temperature detector (RTD) probe block 324 and associated RTD probe 325. The RTD probe 325 is connected by the broken line E to the temperature monitor and controller 703. Line F connects the RTD probe flow block, through the loop circulation pump 316, to the oxygenator 502 which is connected by line G to the bioreactor(s) 201.

As FIG. 3 shows, loop temperature is monitored by an RTD probe 325 inserted into the loop. The RTD probe 325 is coupled to the electronic monitor and controller 703. The controller 703 compares the signal from the RTD probe 325 to a preselected set point and makes adjustments accordingly to a heater 312, preferably mounted externally to a stainless steel portion of the loop.

4. The Basal Medium and Reagent Control System

The basal medium and reagent control system is schematically shown by FIG. 4. Glucose and ammonia analyzer 801 and lactate analyzer 813 are coupled by line H to the basal medium feed pump controller 706 and by line I to the programmable controller 701. The basal medium feed pump controller 706 is coupled by broken line L to the basal medium feed pump 403. The programmable controller 701 is coupled by broken line J to the glucose feed pump 405 and by broken line K to the ECS pump 103.

The glucose and ammonia analyzer 801 is an off line analyzer with multiple dry film based chemistry functions. Samples taken from the loop sample port 314 are manually transferred to the analyzer 801 for glucose and ammonia analysis. Glucose and ammonia assays must be performed independently on the form of analyzer shown.

The off line lactate analyzer 813 consists of a spectrophotometer and analytical means designed specifically for the quantification of lactate through spectrophotometric analysis. Samples taken from the loop sample port 314 are manually transferred to the lactate analyzer 813 for lactate analysis.

Data provided by line H from the analyzers 801 and 813 to the basal feed medium pump controller provides information useful to achieve either manually as shown, or by automation, introduction, of basal medium into the loop at a rate required for a preselected GUR. Preselection of the GUR is based on historical. data obtained with a similar hollow fiber system. Data provided by the line I to the programmable controller 701 achieves introduction of glucose into the loop at a rate required to maintain a specific Go and preselected GUR.

5. The Waste Disposal System

A waste disposal system useful in the invention is schematically shown in FIG. 5.

As shown in FIG. 5, the bioreactor 201 is part of a loop which originates and terminates in the loop reservoir 315. Medium feed line M is connected to the loop line N. Loop pick-up line N extends through the loop circulation pump 316 to the hollow fiber oxygenator 502. Line 0 connects the oxygenator 502 to the hollow fiber reactor 201. As shown, the line L extends into the medium reservoir 315 and terminates at a point above the level of the medium 807 contained therein.

Drain line 803 extends from near the bottom of reservoir 315, through the drain line clamp 804 to a connection with the waste line 806 which extends from the waste exist port 808 at the top of the reservoir 315 and which is provided with waste line clamp 805. A normally closed 0.2 μm filter vent 802 is located on the reservoir above the drain line 803.

A waste displacement system is thus provided, whereby volumes of fresh basal medium entering the loop through line M automatically displace substantially identical volumes of spent medium in the loop through a waste exit port 808 and into waste line 806. Clamp 805 is open during normal operation. Siphoning, caused by venting across the oxygenator membranes, is prevented by the level of medium 807 falling below waste exit port 808 and causing a siphon break.

Reservoir drain line 803 is provided to allow for draining of the reservoir medium. To drain the reservoir 315, the normally closed drain line clamp 804 is opened and the normally open waste line clamp 805 is closed and the normally closed 0.2 μm filter vent 802 is opened. The reservoir can then be drained by siphon.

6. The Oxygenator

FIG. 6 schematically illustrates the hollow fiber oxygenator. As shown in FIG. 6, oxygen depleted medium travels through loop line 306 which is coupled to oxygenator manifold 504. Medium then enters a plurality of hollow fibers 503 formed, e.g., from polyethylene or polypropylene held in place by potting material 510. Gases, usually air, nitrogen and carbon dioxide, are blended, humidified, particulate and sterile filtered prior to entering the ECS 509 of the oxygenator through line 501. Gas exchange occurs across the hollow fiber membranes causing the dissolved gases in the medium to come into equilibrium with the gas mixture introduced into the ECS 509. Oxygen replenished medium exits the hollow fibers 503 and then collects in manifold 505 and is delivered to loop line 307. Depleted ECS gases exit the oxygenator ECS 509 through oxygenator exhaust line 507. The air functions primarily as an oxygen source, nitrogen is useful to reduce oxygen levels and thus ameliorate oxygen toxicity problems and carbon dioxide functions as a pH control agent.

7. The Loop Heater

A preferred form of the loop heater 312 is shown in cross-section by FIG. 7. As illustrated, the heater comprises a stainless steel tube 809, an etched foil resistance heating element 810 and appropriate insulation 811. The heater is provided with suitable wiring 812 and a thermostat 331 as a safety feature. The medium 813 passes through the tube 809 in a flow direction indicated by arrows.

8. Bioreactor System

A preferred form of compact bioreactor system configuration is shown by FIG. 8. In the figure, elements shown in broken line are positioned behind elements shown in solid lines. All elements are directly or indirectly mounted on a transparent polycarbonate or similar sheet.

THE METHOD OF THE INVENTION

The method of the invention is described in general by reference to the figures.

Typical ECS feed material is placed in the ECS feed reservoir 101. As heretofore described, such material may comprise basal medium, growth factors and/or serum.

Typical basal feed material as heretofore described is placed in the basal feed reservoir 402. Such material may comprise basal medium, L-glutamine and antibiotics.

A glucose solution, preferably an aqueous solution contained from about 150 g/l to about 300 g/l glucose, is placed in glucose reservoir 404.

A cell line such as murine hybridoma cell line is propagated in the system as follows: The system excluding the oxygenator 502 and bioreactor 201, which can include multiple bioreactors, as shown in FIGS. 11 and 12, is sterilized by autoclaving. Presterilized oxygenator 502 and bioreactor(s) 201 are incorporated into the loop under a laminar flow hood. Following assembly, the system is placed in a chamber designed specifically to provide system support functions. Electronic cables (broken lines) are provided which connect the loop circulation pump 316, pH probe 332, dissolved oxygen probes 308 and 309, RTD probe 325 and loop heater 312 to the I/O panel of the control chamber. ECS pump 103, basal medium feed pump 403, and glucose feed pump 405 are provided as functional components of the chamber. The oxygenator gas feed line 501 is connected to a chamber port which provides the appropriate premixed gases. Basal medium feed reservoir 402, waste reservoir 401, glucose reservoir 404, ECS feed reservoir 101 and ECS product reservoir 102 having had their contents adjusted appropriately are connected to the system now in place in the chamber.

Once the aforementioned connections have been made the loop section 300 is filled by clamping the loop line 321 downstream of the loop reservoir and activating the basal medium feed pump 403. This action causes the loop to fill in one specific direction and results in a priming of the loop circulation pump 316. The clamp is opened after the loop is completely filled and the loop reservoir 315 is one third filled. At this point the loop circulation pump 316 is activated and the loop circulation flow rate typically set at 350 ml/min per bioreactor for bioreactor(s) with 2.0 m² surface area. Concurrent with this activity the ECS of the bioreactor(s) 201 is filled by manually overriding the ECS control program at programmable controller 701. The ECS pump 103 is activated to pump ECS medium into the bioreactor(s) thereby displacing air from the ECS into the ECS product reservoir 102. This action is continued until the ECS is completely filled with medium. After filling the ECS, the ECS control program is reinstated. The rate of ECS feed and product removal is dependent upon bioreactor surface area, product stability, specific nutrient requirements, and targeted GUR. Maintenance of Go at about one millimolar is appropriate. Simultaneous introduction of growth factor mixture into and removal of product from the ECS at rates that do not differ by more than about five percent is achieved by the programmable controller 701 directing the ECS pump 103. A typical rate of ECS feed and product removal for a murine hybridoma in this system format is 60 ml/day per bioreactor. The ECS product reservoir is collected and replaced as required.

Dissolved oxygen probes 308 and 309 are calibrated by the method described under Detailed Description of Certain System Elements. The DO probes 308 and 309 are calibrated biweekly as part of regular system maintenance. A sample of loop medium is taken from the loop sampler 314 and used to make an off line temperature determination. The temperature controller 703 is adjusted to the off line measured value. At this point the loop heater 312 function is activated by setting the heat control set point. A set point is selected which will maintain a constant system loop temperature of 37° C. Another sample of loop medium is taken from the loop sampler 314 and used to make an off line pH measurement. The pH monitor 704 is then adjusted to be consistent with the off line measured value.

After the aforementioned process parameters have been established the system is allowed a 48 hour check period during which time the probes are allowed to stabilize, system function is verified, and sterility is verified. The basal medium feed pump controller 706 is adjusted to deliver approximately 3 liters of basal medium to the system over the 48 hour check period to serve as a system flush and to remove packing fluids from the bioreactor(s). Upon completion of the 48 hour check period, an inoculum of hybridomas is prepared. The inoculum generally consists of approximately 2.25×10⁸ cells/m² surface area which are suspended in a growth factor mixture which is in turn placed in the ECS feed reservoir 101. The ECS control program is manually overridden to allow for transfer of the inoculum from the ECS feed reservoir 101 through line 104 to the ECS of the bioreactor(s) 201. After completion of the inoculation process, a new ECS feed reservoir 101 containing growth factor mixture is exchanged for the empty inoculation reservoir. A new ECS product reservoir 102 is put in place at this point. Immediately following inoculation, the rate of basal medium delivery to the loop is set by adjusting the basal medium feed pump controller 706 to a value selected on the basis of bioreactor surface area, basal medium type, cell line requirements and targeted GUR. A typical basal medium feed rate for a murine hybridoma with a targeted plateau GUR of 150 mg/hr is 42 ml/hr in bioreactor(s) with 2.0 m² surface area available using Dulbecco's Modified Eagles high glucose medium. Glucose addition as a function separate from basal medium is optional in this system format and requires programming the programmable controller 701 to affect the correct delivery of glucose from the glucose feed reservoir 404 to the loop via the glucose feed pump 405. Typical concentrations of glucose in the glucose feed reservoir 404 range between 150 g/l to 300 g/l. Separation of glucose addition allows for greater control of Go and subsequently greater process control. When glucose is contained in the basal medium, Go values typically settle in a range of 200 mg/l to 300 mg/l. However, when glucose addition is separated from basal medium feed, Go values can be independently controlled at as low as 50 mg/l. Go samples are taken every 24-48 hours from the loop sampler port 314 for off line analysis. Significant plateauing of GUR and .increment.DO values occur approximately 10 days after inoculation. At this point basal medium feed rate and/or glucose feed rate may undergo fine adjustments to help stabilize GURs. See FIG. 9.

After plateauing, GUR in the predescribed format can remain constant without further system adjustments for 65-85 days with diligent system maintenance. .increment.DO values serve as a real time verification of system performance by providing an instantaneous assessment of the metabolic state of the cell mass without performing off line assays and provide a method to instantaneously assess protein production. .increment.DO values are used to monitor the onset of performance problems associated with mass transfer limitations which affect aerobic efficiency. The time of onset of these problems is dependent upon cell type, bioreactor size, and plateau GUR values. The system parameters set forth here would normally incur significant performance loss due to mass transfer limitations at approximately day 70. Aerobic efficiency of the system is determined by the value GUR/.increment.DO where productive plateau values fall in a range of 3-6 (mg/hr)/% air saturation and system cancellation is affected when this value reaches 10-12 (mg/hr)/% air saturation. See FIG. 10.

EXEMPLIFICATION OF THE INVENTION EXAMPLE I

The propagation of an anchorage dependent type cell line in a single bioreactor hollow fiber cell propagation system of the invention is described in this example. The cell line is a Crandall feline kidney (CRFK) Dixon strain fibroblast that persistently sheds feline immunodeficiency virus (FIV) and associated proteins. The product of interest in the culture supernatant is a viral core glycoprotein p28, quantified by enzyme linked immunoadsorbent assay (ELISA).

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as generally described in "The Method of the Invention" section.

The process parameters in the culture system were set to establish start up conditions. The set point for the temperature controller 703 was set at 37° C. The gas flow meters 609 were adjusted such that the pH monitor 704 read 7.25, and the DO monitors 702 read about 70% air saturation. The loop circulation controller 705 was set to obtain a loop circulation rate of 400 ml/min. The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 50 ml/day.

An inoculum containing 4.5×10⁸ cells was pumped into the ECS of the bioreactor 201 as generally described in "The Method of the Invention" section. A 2.0 m² surface area bioreactor (model FC-20, Toray Industries) 201 was used in this run. The growth factor mixture comprised 40% fetal bovine serum, 2% L-glutamine, 1% penicillin streptomycin, in Dulbecco's Modified Eagle's (DME) high glucose (4500 mg/l) basal medium. This composition was maintained throughout the run. The ECS feed reservoir 101 and ECS product reservoir 102 were replaced three times/week throughout the run.

The basal medium feed pump controller 706 was set to deliver 41 ml/hr. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME high glucose basal medium. This composition was maintained throughout the run. Thus, the optional separate glucose feed system was not used in this run. The basal medium feed reservoir 402 was replaced 3 times/week, and the waste reservoir 401 was replaced when necessary.

Samples were withdrawn regularly for off line glucose analysis, as generally described in "The Method of the Invention" section. As Table 1 shows, the glucose utilization rate (GUR) and the dissolved oxygen differential across the bioreactor (.increment.DO) both increased as the cells proliferated in the ECS of the bioreactor 201.

                  TABLE 1                                                          ______________________________________                                         Day #   GUR (mg/hr)   ΔDO (%)                                                                           p28 (μg/day)                                 ______________________________________                                         1       10.000                                                                 2       15.000                                                                 3       15.600                                                                 5                                                                              6       32.700                                                                 7       28.500        0.300                                                    8       41.700        0.600    150.000                                         9       44.200        1.500                                                    10      48.100        2.200    100.000                                         11                                                                             12                                                                             13      51.280                 350.000                                         14      50.600                                                                 15      47.200        9.900    350.000                                         16      47.200        13.500                                                   17      52.400        14.100   375.000                                         18                                                                             19                    16.000                                                   20      59.500        15.800   500.000                                         21      66.000        14.800                                                   22      61.500        13.800   300.000                                         23      63.500        14.400                                                   24      67.300        14.200   300.000                                         25      67.900        15.600                                                   26                                                                             27                    23.800                                                   28      71.800        21.100   360.000                                         29      60.300        22.400                                                   30      51.900        22.400                                                   31      55.100        21.400   292.000                                         ______________________________________                                    

The target GUR was set at 60 mg/h. By day 13 the GUR and p28 protein production stabilized significantly. The production phase that followed was maintained for almost three weeks. The GUR, p28 production and .increment.DO profiles were all relatively stable and parallel during this period.

The goals of this run were to demonstrate an ability to culture this anchorage dependent cell line in the invention, and to manufacture enough viral p28 protein to meet in house research requirements. Having met these objectives, the run was terminated.

EXAMPLE II

The propagation of a suspension type cell line in a single bioreactor hollow fiber cell propagation system of the invention is described in this example. The cell line is a murine hybridoma that secretes monoclonal antibodies against transmissible gastro enteritis virus (TGEV). The IgG_(2a) antibody product in the culture supernatant is measured by radial immuno diffusion (RID) assay.

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as generally described in "The Method of the Invention" section.

The process parameters in the culture system were set to establish start up conditions. The set point for the temperature controller 703 was set at 37° C. The gas flow meters 609 were adjusted such that the pH monitor 704 read 7.31, and the DO monitors 702 read about 95% air saturation. Nitrogen flowrate in this run was set to zero. The loop circulation controller 705 was set to obtain a loop circulation rate of 350 ml/min. The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 24 ml/day.

An inoculum containing 3.1×10⁸ cells was pumped into the ECS of the bioreactor 201 as generally described in "The Method of the Invention" section. A 2.0 m² surface area bioreactor (model FC-20, Toray Industries) 201 was used in this run. The growth factor mixture comprised 35% fetal bovine serum, 1% penicillin streptomycin, in DME high glucose basal medium. This composition was maintained throughout the run. The ECS feed reservoir 101 and ECS product reservoir 102 were replaced three times/week throughout the run.

The basal medium feed pump controller 706 was set to deliver 50.4 ml/hr. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME high glucose basal medium. This composition was maintained throughout the run. Thus, the optional separate glucose feed system was not used in this run. The basal medium feed reservoir 402 was replaced 3 times/week, and the waste reservoir 401 was replaced when necessary.

Samples were withdrawn regularly for off line glucose analysis, as generally described in "The Method of the Invention" section. As Table 2 shows, the GUR and .increment.DO both increased as the cells proliferated in the ECS of the bioreactor 201.

                  TABLE 2                                                          ______________________________________                                         Day #   GUR (mg/hr)   ΔDO (%)                                                                           Ab (mg/day)                                     ______________________________________                                         1       9.570                                                                  3       37.200                 1.920                                           4       50.900                                                                 5       66.000                 8.640                                           6       94.460                                                                 7       110.750       14.000   32.160                                          8                                                                              9                                                                              10      155.700       11.600   47.520                                          11      148.800       15.200                                                   12      161.200       13.300   64.320                                          13      149.800       13.200                                                   14      145.500       12.100   90.480                                          15                                                                             16                                                                             17      137.500       11.300                                                   18      143.900                124.560                                         19                                                                             20      168.600                                                                21      168.000       26.400   135.120                                         22                                                                             23                                                                             24      115.600       24.700                                                   25      127.450       33.500   135.120                                         26      142.200       34.100                                                   27      153.180       33.100                                                   28      156.900       27.200   140.400                                         29                                                                             30                                                                             31      155.290       38.600   151.440                                         32      145.600       34.300                                                   33      145.600       34.300   156.960                                         34      146.000       28.500                                                   35      146.850       30.900   162.720                                         36                                                                             37                                                                             38      147.700       32.800   168.480                                         39      147.200                                                                40      148.100       34.100   204.936                                         41      149.380       31.900                                                   42      149.380       29.400   189.600                                         43                                                                             44                                                                             45      151.500       32.000   194.664                                         46      151.500       27.100                                                   47      151.500       35.400   179.736                                         48      153.100       32.100                                                   49      153.300       29.400   179.736                                         ______________________________________                                    

The basal medium feed rate was manipulated to establish and maintain a target GUR of 150 mg/hr. By day 10 the GUR stabilized significantly, and by day 22, the antibody production [Ab} and .increment.DO stabilized too. The production phase following immediately was maintained for four weeks, during which, the antibody production to .increment.DO ratio exhibited relative constancy.

The goals of this run were to demonstrate an ability to culture this suspension type cell in the invention, and to manufacture enough antibody to meet in house research requirements. Having met these objectives, the run was terminated.

EXAMPLE III

The propagation of a suspension type cell line in a single bioreactor hollow fiber cell propagation system of the invention is described in this example to illustrate improved productivity by means of controlling the effects of product and metabolite feedback inhibition. The cell line is a murine hybridoma that secretes monoclonal antibodies against TGEV. The IgG_(2a) antibody product in the culture supernatant is measured by RID assay.

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as generally described in "The Method of the Invention" section.

The process parameters in the culture system were set to establish start up conditions. The set point for the temperature controller 703 was set at 37° C. The gas flow meters 609 were adjusted such that the pH monitor 704 read 7.3. Nitrogen flowrate in this run was set to zero. DO monitors 702 were excluded in this run. The loop circulation controller 705 was set to obtain a loop circulation rate of 350 ml/min. The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 24 ml/day. This volumetric feed rate was varied through the run as described below.

An inoculum containing 3×10⁸ cells was pumped into the ECS of the bioreactor as generally described in "The Method of the Invention" section. A 1.4 m² surface area bioreactor (model 1-L, C.D. Medical Inc.) 201 was used in this run. The growth factor mixture comprised 35% fetal bovine serum, 1% penicillin streptomycin, in DME high glucose basal medium. This composition was varied through the run as described below. The ECS feed reservoir 101 and ECS product reservoir 102 were replaced three times/week throughout the run.

The basal medium feed pump controller 706 was set to deliver 41.5 ml/hr. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME high glucose basal medium. This composition was maintained throughout the run. Thus, the optional separate glucose feed system was not used in this run. The basal medium feed reservoir 402 was replaced 3 times/week, and the waste reservoir 401 was replaced when necessary.

Samples were withdrawn regularly for off line glucose analysis, as generally described in "The Method of the Invention" section. As Table 3(a) shows, the GUR and .increment.DO both increased as the cells proliferated in the ECS of the bioreactor 201.

                  TABLE 3(a)                                                       ______________________________________                                         Day #   GUR (mg/hr)  ECS (ml/day)                                                                              Ab (mg/day)                                    ______________________________________                                         1       12.0         24                                                        2       31.9         24                                                        3                    24                                                        4       45.2         24         17.28                                          5       52.7         24                                                        6       53.5         24         50.40                                          7       83.8         24                                                        8       100.4        24         43.68                                          9       91.7         24                                                        10      100.0        24                                                        11      103.8        24         80.16                                          12      84.2         24                                                        13      88.5         24         88.32                                          14      82.9         24                                                        15      80.8         24         88.32                                          16                   24                                                        17                   24                                                        18      81.2         24         88.32                                          19      85.7         24                                                        20      100.1        24         88.32                                          21      105.2        24                                                        22      109.7        24         96.48                                          23                   24                                                        24      105.8        24                                                        25                   24                                                        26      104.0        24         121.44                                         27      102.1        30                                                        28      103.4        30                                                        29      103.1        30         151.80                                         30                   30                                                        31                   30                                                        32      109.1        30         151.80                                         33      108.6        30                                                        34                   30         130.80                                         35      110.7        42                                                        36      112.8        42         168.84                                         37                   42                                                        38                   42                                                        39      114.6        42         168.84                                         40      114.6        42                                                        41      114.6        42         154.56                                         42                   42                                                        43      113.1        42         153.72                                         44                   54                                                        45                   54                                                        46      114.3        54         163.08                                         47      113.7        54                                                        48      113.7        54         163.08                                         49      114.6        54                                                        50      114.6        54         163.08                                         51                   60                                                        52                   60                                                        53      114.9        60         162.00                                         54      115.5        60                                                        55      115.5        60         162.00                                         ______________________________________                                    

The basal medium feed rate was manipulated to establish and maintain a target GUR of 100 mg/hr. By day 8 the GUR stabilized significantly, and by day 26, the antibody production stabilized too.

The production phase following immediately was maintained for almost four weeks. During this period, the ECS volumetric feed rate (and ECS product collection rate) was maintained at 30, 42 and 54 ml/day, each for at least a week. The fetal bovine serum content of the growth factor mixture was adjusted to ensure constant serum feed to the system. As Table 3(b) shows, while the increase in ECS feed rate had no effect upon GUR, it improved the antibody productivity by 45%.

                  TABLE 3(b)                                                       ______________________________________                                         ECS (ml/day)    Ab (mg/day)                                                    ______________________________________                                         OLDER RUN                                                                      24.000          112.800                                                        24.000          96.000                                                         24.000          108.000                                                        36.000          162.000                                                        36.000          154.800                                                        36.000          144.000                                                        36.000          144.000                                                        36.000          144.000                                                        48.000          139.200                                                        CURRENT RUN                                                                    24.000          96.480                                                         24.000          121.440                                                        30.000          151.800                                                        30.000          151.800                                                        30.000          130.800                                                        42.000          168.840                                                        42.000          168.840                                                        42.000          154.560                                                        42.000          153.720                                                        54.000          163.080                                                        54.000          163.080                                                        54.000          163.080                                                        60.000          162.000                                                        60.000          162.000                                                        ______________________________________                                    

Thus, the effects of product and metabolite feedback inhibition were overcome by maintaining a higher ECS product collection rate (or ECS feed rate). The combined results of this exercise and that from another run are reported in Table 3(b). It was concluded that for the given set of process conditions, the ECS feed rate must be set to at least 42 ml/day to maximize antibody productivity.

The goal of this run was to illustrate improved productivity by means of controlling the effects of product and metabolite feedback inhibition. Having met this objective, the run was terminated.

EXAMPLE IV

The propagation of a suspension type cell line in a single bioreactor hollow fiber cell propagation system of the invention is described in this example to illustrate improved process control by means of an optional separate glucose feed system. The cell line is a murine hybridoma that secretes monoclonal antibodies against TGEV. The IgG_(2a) antibody product in the culture supernatant is measured by RID assay.

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as generally described in "The Method of the Invention" section.

The process parameters in the culture system were set to establish start up conditions. The set point for the temperature controller 703 was set at 37° C. The gas flow meters 609 were adjusted such that the pH monitor 704 read 7.2, and the DO monitors 702 read about 95% air saturation. Nitrogen flowrate in this run was set to zero. The loop circulation controller 705 was set to obtain a loop circulation rate of 350 ml/min. The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 35 ml/day.

An inoculum containing 3×10⁸ cells was pumped into the ECS of the bioreactor 201 as generally described in "The Method of the Invention" section. A 2.0 m² surface area bioreactor (model FC-20, Toray Industries) was used in this run. The growth factor mixture comprised 24% fetal bovine serum, 1% penicillin streptomycin, in DME glucose free basal medium. This ECS feed composition was maintained throughout the run. The ECS feed reservoir 101 and ECS product reservoir 102 were replaced three times/week throughout the run.

The basal medium feed pump controller 706 was set to deliver 42 ml/hr. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME glucose free basal medium. This composition was maintained throughout the run. The programmable controller 701 was set to deliver glucose, via the glucose feed pump 405, such that the effective glucose concentration in the feed medium (Gi) was 1198 mg/l. The glucose reservoir 404 contained glucose stock solution at 200 g/l. The basal medium feed reservoir 402 was replaced 3 times/week, the glucose reservoir was replaced 1 time/week, and the waste reservoir 401 was replaced when necessary.

Samples were withdrawn regularly for off line glucose analysis, as generally described in "The Method of the Invention" section. As shown in Table 4 below, the Gi was manipulated via the programmable controller 701, to support the increasing GUR and .increment.DO of the proliferating cells in the bioreactor 201, and at the same time maintain an approximately constant system glucose concentration (Go) of 800 mg/l.

                  TABLE 4                                                          ______________________________________                                         Day                                                                            #    Gi (mg/l)                                                                               Go (mg/l)                                                                               GUR (mg/hr)                                                                             ΔDO (%)                                                                         Ab (mg/day)                             ______________________________________                                         1    1198     790.0    17.1                                                    2    2001     770.0    51.6     6.2                                            3    2489     840.0    69.3     9.6                                            4    2716     840.0    79.0     11.2                                           5    2845     830.0    84.8     12.1                                           6    2923     830.0    88.1     13.7                                           7    2975     780.0    92.4     16.1   40.950                                  8    3148     840.0    97.4     18.0                                           9    2606     360.0    94.3     18.6   62.300                                  10   2489     270.0    93.2     21.0                                           11                                                                             12   2489     240.0    94.5     22.6   82.950                                  13   2489     210.0    95.7     25.1                                           14   2489     157.0    97.9     29.6   93.100                                  15   2489     123.0    99.4     33.1                                           16   2489     107.0    100.0    33.5   115.150                                 17                                                                             18   2489     86.0     100.9    35.1                                           19   2489     86.0     100.9    35.6   127.050                                 20   2489     80.0     101.2    37.1                                           21   2489     68.0     101.7    38.1   133.000                                 22   2489     68.0     101.7    38.6                                           23   2489     51.0     102.4    40.6   133.000                                 24                                                                             25                                                                             26   2489     49.0     102.5    41.0   144.900                                 27   2489     53.0     102.3    43.0                                           28   2489     48.0     102.5    44.1   148.750                                 29   2489     50.0     102.4                                                   30   2489     48.0     102.5    41.6   154.000                                 31                                                                             32                                                                             33   2489     47.0     102.5    44.6   154.000                                 34   2489     51.0     102.4    44.7                                           35   2489     49.0     102.5    45.1   148.750                                 ______________________________________                                    

By day 8 the GUR reached a target value of 100 mg/hr, at which point, the glucose pump was momentarily shut off to rapidly bring Go down to 300 mg/l. Then, the programmable controller 701 was reset to obtain a Gi of 2489 mg/l, to support the established of 100 mg/hr, and at the same time allow the Go to settle below 1 mM (or 180 mg/l).

The basal medium feed pump controller 706 was set to deliver 42 ml/hr from the beginning of the run, a basal medium feed rate that can support a GUR of 150 mg/hr. However, immediately following the Go manipulation on day 8, the GUR levelled at 100 mg/hr, and .increment.DO at 22%, as a result of glucose limitation (200 mg/l <Go<300 mg/l). Thus, a separate glucose feed system made possible an independent and useful control of the process. After day 14, further glucose limitation (Go<180 mg/l) led to an increasingly aerobic GUR as a result of the Pasteur shift in cell metabolism. Thus, over the next three weeks, although the GUR remained relatively unchanged at 100 mg/hr, the .increment.DO eventually stabilized at 44%. The antibody production stabilized at 154 mg/hr, which is comparable to production values obtained under similar process conditions but non limiting glucose values (Go>300 mg/l), as seen in Example 3.

The goal of this run was to demonstrate an improved ability to control the process by means of using a separate glucose feed system. Having met this objective, the run was terminated at this point.

EXAMPLE V

The propagation of an anchorage dependent cell line in both single and multiple bioreactor hollow fiber cell propagation systems of the invention is described in this example to illustrate scale-up of protein production. A single bioreactor system and a four bioreactor system were each run using a recombinant chinese hamster ovary (rCHO) cell line. The product of interest in the culture supernatant is a 70-90 Kd proteoglycans, a glycoprotein, quantified by ELISA.

Single Reactor Run

A single 2.0 m² surface area bioreactor (model FC-20, Toray Industries) 201 was used for this run. The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as described in "The Method of the Invention" section. The process parameters in the culture system were set to establish start up conditions. The set point for the temperature controller 703 was set to 37° C. The gas flowmeters 609 were adjusted such that the pH monitor 704 read 7.26, and the DO monitors read about 65% air saturation. The loop circulation controller 705 was set to obtain a loop circulation rate of 400 ml/min. The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 50 ml/day.

An inoculum containing 4.5×10⁸ cells was pumped into the ECS of the bioreactor 201 as described in "The Method of the Invention" section. The growth factor mixture comprised 40% fetal bovine serum, 1% penicillin streptomycin, in Dulbecco's Modified Eagle's (DME) high glucose (4500 mg/l) basal medium. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, 1% non-essential amino acids (NEAA) in DME high glucose basal medium. The basal medium feed pump controller 706 was set to deliver 40 ml/hr. Production rate of proteoglycans was relatively stable for seven weeks at an average of 141 μg/day.

Four Reactor Run

Scale-up was accomplished by using four 2.0 m² surface area bioreactors (model FC-20, Toray Industries) 201 in a multiple bioreactor configuration as described in "The System of the Invention" section. The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as described in "The Method of the Invention" section.

The temperature, pH and DO were set to duplicate conditions in the single bioreactor run. The loop circulation controller 705 was set to obtain a loop circulation rate of 1400 ml/min (350 ml/min per bioreactor). The programmable controller 701 was set to deliver bioreactor ECS feed medium (and collect ECS product) at a frequency of 24 times/day and a volume of 256 ml/day (64 ml/day per bioreactor).

An inoculum containing 1.8×10⁹ (4.5×10⁸ per bioreactor) cells was pumped into the ECS of the bioreactors 201 as described in "The Method of the Invention" section. The growth factor mixture comprised 40% fetal bovine serum, 1% penicillin streptomycin in Dulbecco's Modified Eagle's (DME) high glucose (4500 mg/l) basal medium. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, 1% NEAA in DME high glucose basal medium. The basal medium feed pump controller 706 was set to deliver 166.7 ml/hr (41.6 ml/hr per bioreactor). Production rate of proteoglycans averaged 870 μg/day over a three week period.

The scale-up factor for protein production was calculated at 6.2. On a per bioreactor basis, an ECS feed rate (and product collection rate) of 64 ml/day was used for the four bioreactor run as opposed to 40 ml/day in the single bioreactor run. The concentrations of proteoglycans in both runs were comparable.

EXAMPLE VI

The propagation of an anchorage dependent cell line in a multiple bioreactor hollow fiber cell propagation system of the invention is described in this example to illustrate scale-up of protein production. Scale-up was accomplished by using four 2.0 m² surface area bioreactors (model FC-20, Toray Industries) 201 in a multiple bioreactor configuration as described in "The System of the Invention" section. The cell line is a CRFK Dixon strain fibroblast that persistently sheds FIV and associated proteins, as described in Example I above. The product of interest in the culture supernatant is a viral core glycoprotein p28, quantified by ELISA.

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as described in "The Method of the Invention" section. The temperature, pH and DO were set to duplicate conditions as used for Example I. The loop circulation controller 705 was set to obtain a loop circulation rate of 1400 ml/min (350 ml/min per bioreactor). The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 200 ml/day (50 ml/day per bioreactor).

An inoculum containing 1.09×10⁹ (2.6×10⁸ per bioreactor) cells was pumped into the ECS of the bioreactors 201 as described in "The Method of the Invention" section. The growth factor mixture comprised 25% fetal bovine serum, 2% L-glutamine, 1% penicillin streptomycin, in Dulbecco's Modified Eagle's (DME) high glucose (4500 mg/l) basal medium. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME high glucose basal medium. The basal medium feed pump controller 706 was manipulated and eventually set to deliver 166 ml/hr (41.5 ml/hr bioreactor). This basal medium feed rate corresponds to a scale-up factor of 4 over that used in the single bioreactor system in Example 1. Production rates of p28 protein were relatively stable for six weeks, and reached up to 1208 μg/day. An average production rate of 366 μg/day was seen during the production phase in Example 1. Thus, the production scale-up factor for p28 protein was 3.3.

EXAMPLE VII

The propagation of a suspension type cell line in a multiple bioreactor hollow fiber cell propagation system of the invention is described in this example. The express purpose of this run was to test the ability of the lumenal and ECS manifold system to deliver nutrients evenly to each of the bioreactors in a multiple bioreactor configuration, rather than a demonstration of scale-up of protein production. One system having two banks of four bioreactors was used, each bank containing four 2.0 m² surface area bioreactors (model FC-20, Toray Industries) 201 in a configuration as described in "The System of the Invention" section. The cell line was a murine hybridoma that secretes monoclonal antibodies against TGEV, as described in Example II above. The IgG_(2a) antibody product in the culture supernatant is measured by RID assay.

The culture system was sterilized, assembled, connected to accessories and chamber controls, primed and flushed, as described in "The Method of the Invention" section. The temperature, pH and DO were set to duplicate conditions in Example II. The loop circulation controller 705 was set to obtain a loop circulation rate of 2000 ml/min (250 ml/min per bioreactor). The programmable controller 701 was set to deliver growth factor mixture (and collect ECS product) at a frequency of 6 times/day and a volume of 288 ml/day (36 ml/day per bioreactor).

An inoculum containing 3.2×10⁹ (4.0×10⁸ per bioreactor) cells was pumped into the ECS of the bioreactors 201 as described in "The Method of the Invention" section. The growth factor mixture comprised 40% fetal bovine serum, 2% L-glutamine, 1% penicillin streptomycin, in Dulbecco's Modified Eagle's (DME) high glucose (4500 mg/l) basal medium. The basal feed medium comprised 2% L-glutamine, 1% penicillin streptomycin, in DME high glucose basal medium. The basal medium feed pump controller 706 was set to deliver 333 ml/hr (41.6 ml/hr per bioreactor).

On day 15, the ECS of each bioreactor was sampled individually and assayed for antibody concentration. In the first multiple bioreactor set, the antibody concentration was 2.33±0.21 mg/ml. In the second multiple bioreactor set, the antibody concentration was 1.83±0.21 mg/ml. Thus, the percentage variation in antibody concentration for the first and second set is 6.55% and 11.24%, respectively. This experimental result essentially represents an identical product concentration in different bioreactors in the set. The premise is that in each multiple bioreactor set, an equal concentration of product in each bioreactor means that there must be even cell growth in each bioreactor, which is possible only if there is an even distribution of nutrients to each bioreactor 201. Thus, the functional efficacy of the lumenal and ECS manifold system was demonstrated. 

We claim:
 1. A cell propagation system comprising:(i) at least two hollow fiber bioreactors each of said bioreactors comprising:a casing, hollow fibers positioned in said casing, an extracapillary space defined by the interior surface of said casing and the exterior surfaces of said hollow fibers, a first inlet means for fluid flow into the lumens of said hollow fibers, a second inlet means for fluid flow into said extracapillary space, said second inlet means further comprising valve means for the control of fluid separately through said extracapillary space of each of said bioreactors, a first outlet means for withdrawal of fluid from the lumens of said hollow fibers, a second outlet means for withdrawal of fluid from said intracapillary space, (ii) a fluid flow path connecting said first inlet and said first outlet means of each of said bioreactors, (iii) a fluid flow path connecting said second inlet means and said second outlet means of each of said bioreactors, and (iv) programmable controller means for activating and deactivating said valve means comprised by each of said second inlet means of each of said bioreactors.
 2. The cell propagation system of claim 1 wherein said programmable controller means (iv) is programmed to activate and deactivate each of said valve means sequentially.
 3. The cell propagation system of claim 1 or claim 2 further comprising:(v) manifold means connecting said second inlet means and said second outlet means of each of said bioreactors.
 4. The cell propagation system of claim 1 in which said second inlet means and said second outlet means provide single stream fluid entry into said extracapillary space and single stream withdrawal of fluid from said extracapillary space of each of said at least two hollow fiber bioreactors.
 5. The cell propagation system of claim 1 further comprising means for providing a controlled fluid flow rate through said fluid flow path connecting said second inlet means and said second outlet means of each of said bioreactors.
 6. The cell propagation system of claim 1 further comprising means for introduction of basal medium and nutrients into said fluid flow path (ii) at a rate determined by a preselected nutrient utilization rate in said bioreactors.
 7. A hollow fiber cell propagation system comprising:(i) at least two hollow fiber bioreactors including nutrient permeable hollow fibers positioned in extracapillary space of said bioreactors; (ii) a loop for controlling the flow of fluids through the lumens of said fibers, said loop comprising:(a) pump means for circulating fluids through said loop; (b) means for introducing gases into said loop; means for determining and controlling the temperature of said fluids; (d) means for determining and controlling the dissolved oxygen content of said fluids upstream or downstream of said bioreactors; (e) means for determining and controlling the flow rate of said fluids through said loop; (f) means for determining and controlling pH of said fluids; (g) means for introducing fluids comprising basal medium containing said nutrient into said loop; and (iii) means for controlling the flow of fluids through said extracapillary spaces; wherein said loop further comprises dissolved oxygen determining means for measuring dissolved oxygen content of fluids in said loop, said dissolved oxygen determining means of paragraph (ii)(d) positioned in flow block means with a sensing portion of said dissolved oxygen determining means positioned in said flow block means so as to be in contact with fluid flowing in said loop, said flow block means including sterile filter means for passing calibrating gas into said flow block means so as to be in contact with said sensing portion for calibrating said dissolved oxygen determining means at a pressure equal to that of fluid flowing in said loop.
 8. A process for the conversion of a raw material to a useful product by cell metabolism which comprises(i) inoculating the extracapillary space of at least two hollow fiber bioreactors with cells and a growth factor mixture for said cells; (ii) circulating basal medium comprising a nutrient into and through the lumens of the hollow fibers of said bioreactors; (iii) introducing oxygen into said circulating basal medium to establish and maintain an oxygen content sufficient to support aerobic metabolism of said cells; (iv) utilizing the differential in dissolved oxygen content of the circulating basal medium before and after passage through the lumens of the hollow fibers of said bioreactors to provide a real time verification of cell metabolism; wherein the dissolved oxygen content of the circulating basal medium is measured using a dissolved oxygen determining means that is calibrated using the following steps:positioning said dissolved oxygen determining means in a flow block means with a sensing portion of said dissolved oxygen determining means positioned in said flow block means so as to be in contact with circulating basal medium; passing calibrating gas into said flow block means through a sterile filter means so as to be in contact with said sensing portion for calibrating said dissolved oxygen determining means at a pressure equal to that of circulating basal medium.
 9. A hollow fiber cell propagation system comprising:(i) at least two hollow fiber bioreactors each of said bioreactors comprising:a casing, hollow fibers positioned within said casing, and an extracapillary space defined by the interior surface of said casing and the exterior surfaces of said hollow fibers, (ii) a first fluid flow loopsaid first fluid flow loop being connected to first inlet means and to first outlet means to provide flow of fluids through the lumens of said hollow fibers, (iii) a fluid flow path connected to second inlet means and second outlet means to provide flow of fluids through said extracapillary space,said fluid flow path further comprising controller means for providing a controlled fluid flow rate for introducing fluids into and removal of product from said extracapillary spaces of each of said bioreactors, with fluid flow through each of said bioreactors controlled by valve means, wherein said valve means are directed to open or close in response to signals from said controller means so as to permit said fluid flow only through the extracapillary space of one of said bioreactors at a time.
 10. A process for the conversion of a raw material to a useful product by cell metabolism which comprises inoculating extracapillary spaces of at least two hollow fiber bioreactors in which said inoculation of cells and growth factor mixture into said extracapillary space comprises controlled fluid flow for introduction of fluids into and removal of product from said extracapillary space of each of said bioreactors with said fluid flow permitted through only one of said bioreactors at a time.
 11. The claim 10 process in which equal transmembrane pressure is maintained in each of said at least two hollow fiber bioreactors. 